This invention relates to devices for molecular synthesis, storage and screening, and other chemical, biochemical, biological, and physical experiments, and to methods of making, using, and manipulating the same.
High throughput methods for creating and analyzing chemical and biochemical diversity play a vital role in technologies including drug discovery and development. Specific applications of high throughput methods include drug discovery, optimization of reaction conditions (e.g., conditions suitable for protein crystallization), genomics, proteomics, genotyping, polymorphism analysis, examination of RNA expression profiles in cells or tissues, sequencing by hybridization, and recombinant enzyme discovery.
Rapid, high throughput methods for synthesizing (e.g., using combinatorial chemistry methods) and screening large numbers of these compounds for biological and physicochemical properties are desired, for example, to increase the speed of discovery and optimization of drug leads.
Similarly, due in part to the large amount of sequence data from the human genome project, efforts are underway to rapidly obtain x-ray crystallography data for the protein products of many newly discovered genes. One of the rate limiting steps in this process is the search for appropriate solution conditions (e.g., pH, salt concentration) to cause protein crystallization. There is also a need to determine the function of each of the newly discovered genes (i.e., xe2x80x9cfunctional genomicsxe2x80x9d) and to map protein-protein interactions (i.e., xe2x80x9cproteomicsxe2x80x9d). Given the large number of human genes, protein modifications, and protein binding partners, higher throughput methods are desired.
Another advance in biotechnology is the creation of surfaces with high-density arrays of biopolymers such as oligonucleotides or peptides. High-density oligonucleotide arrays are used, for example, in genotyping, polymorphism analysis, examination of RNA expression profiles in cells or tissues, and hybridization-based sequencing methods as described, for example, in U.S. Pat. Nos. 5,492,806, 5,525,464, and 5,667,972 to Hyseq, Inc. Arrays containing a greater number of probes than currently provided are desirable.
The process of discovering and improving recombinant enzymes for industrial or consumer use has emerged as an important economic activity in recent years. A desire to discover very rare, activity-improving mutations has further stimulated the search for higher throughput screening methods. Such methods often require screening 100,000 to 1,000,000 members of a genetic library in parallel, and then rapidly detecting and isolating promising members for further analysis and optimization.
One of the challenges in the development of high throughput methods is that conventional liquid handling techniques such as pipetting, piezoelectric droplet dispensing, split pin dispensing, and microspritzing are generally unsuitable for rapidly loading or transferring liquids to or from plates of high density (e.g., plates having more than about 384 wells). For example, these techniques can cause substantial splashing, resulting, for example, in contamination of neighboring wells and loss of sample volume. Also, as the number of wells increases, the time necessary to reformat compounds from the previous generation of plates to the higher density plates generally increases, thus limiting the utility of higher density plates. Evaporation can also be problematic with times greater than a few seconds. Moreover, entrapped air bubbles can result in inconsistencies in the loading of small fluid volumes (e.g., less than about one microliter).
Significant bottlenecks in high throughput screening efforts include library storage, handling, and shipping. As the number of compounds in a library increases, the number of 96- or 384-well plates, and the total volume needed to store the libraries, also increases. For compounds that are stored in frozen solvent such as DMSO or water, thawing, dispensing, and refreezing pose the hazard of crystallization, precipitation, or degradation of some compounds, making it difficult to dispense accurate quantities in the future. Having samples stored in low-density plates requires a time consuming step of reformatting the samples into high-density plates before the high-density technology can be utilized.
The invention features methods of making devices, or xe2x80x9cplatensxe2x80x9d, having a high-density array of through-holes, as well as methods of cleaning and refurbishing the surfaces of the platens. The invention further features methods of making high-density arrays of chemical, biochemical, and biological compounds, having many advantages over conventional, lower-density arrays. The invention includes methods by which many physical, chemical or biological transformations can be implemented in serial or in parallel within each addressable through-hole of the devices. Additionally, the invention includes methods of analyzing the contents of the array, including assaying of physical properties of the samples.
In various embodiments, the reagents can be contained within the through-holes by capillary action, attached to the walls of the through-holes, or attached to or contained within a porous material inside the through-hole. The porous material can be, for example, a gel, a bead, sintered glass, or particulate matter, or can be the inner wall of a through-hole that has been chemically etched. In particular embodiments, the arrays can include individual molecules, complexes of molecules, viruses, cells, groups of cells, pieces of tissue, or small particles or beads. The members of the arrays can also, for example, function as transducers that report the presence of an analyte (e.g., by providing an easily detected signal), or they can function as selective binding agents for the retention of analytes of interest. Using these methods, arrays corresponding to a large plurality of human genes (e.g., using nucleic acid probes) can also be prepared.
On embodiment of the invention features a method of making a platen of a desired thickness having a plurality of through-holes. The method includes the steps of (a) providing a plurality (e.g., 2, 3, 5, 8, 10, 100, 1000 or more) of plates having upper and lower surfaces, wherein one or both of the upper and lower surfaces of at least some of said plurality of plates has continuous, substantially parallel grooves running the length of said surfaces; (b) bonding the upper surfaces of all but one of said plurality of plates to the lower surfaces of the other plates (i.e., the upper surface of the first plate is bonding to the lower surface of the second plate; the upper surface of the second plate is bonded to the lower surface of the third plate; and so on; the upper surface of the last plate is not bonded to anything else); and (c) if necessary to achieve the desired thickness, slicing the platen substantially perpendicularly to the through-holes, thereby creating a platen of a desired thickness having a plurality of through-holes. Step c) can optionally be repeated make a plurality of platens.
The plates can be made from any material that can be bonded (e.g., plastic, metal, glass, or ceramic), and each can have a thickness from, e.g., about 0.01 mm to 2.0 mm, preferably 0.1 mm to 1 mm; the grooves have a depth from, e.g., 0.005 mm to 2.0 mm (i.e., less than the thickness of the plates); and the grooves can have a width from, e.g., 0.1 mm to 1.0 mm.
The plates can be bonded in a configuration in which the grooves of one plate are substantially parallel to the grooves of each of the other plates, or can be bonded so that the grooves of certain plates are perpendicular to, or at acute angles to, the grooves of certain other plates.
In another embodiment, the invention features a device for the immobilization of probes, cells, or solvent. The device includes a platen (optionally having hydrophobic upper and lower surfaces) having a plurality of through-holes (e.g., from the upper surface to the lower surface), where at least some of the through-holes contain a porous material such as a gel (e.g., polyacrylamide), silica, sintered glass, or polymers for the immobilization of probes, cells, or solvent.
In still another embodiment, the invention features a method of making a platen having opposing hydrophobic surfaces and a plurality of hydrophilic through-holes. The method includes the steps of: (a) coating a plate with a material (e.g., gold, silver, copper, gallium arsenide. metal oxides, or alumina) that reacts with amphiphilic molecules (e.g., alkane thiols, alkanephosphates, alkane carboxylates); (b) forming through-holes in the plate (e.g., by micromachining methods such as drilling, electrospark discharge machining (EDM), punching, stamping, or etching; and (c) treating (e.g., dipping or spraying) the plate with a solution or vapor of an amphiphilic molecule to provide a platen having hydrophobic coating on surfaces of the platen but not on the walls of the through-holes. The invention also includes the platens made by this method, as well as a method of regenerating the hydrophobic coating on the platen after use. This method includes the steps of: (a) removing residual hydrophobic coating, if any (e.g., by washing the platen with oxidant, reductant, acid, base, or detergent, or by heating, electropolishing, irradiating, or burning); and (b) treating the platen with a solution or vapor of an amphiphilic molecule to regenerate the hydrophobic coating.
In yet another embodiment, the invention features a method of selectively making a coating on the surfaces of a platen having a plurality of through-holes. The method includes the steps of: (a) selectively coating the surfaces of the platen with a material that reacts with amphiphilic molecules; and (b) treating the platen with a solution or vapor of an amphiphilic molecule to regenerate the hydrophobic coating.
Still another embodiment of the invention features a platen having two opposing surfaces and a plurality of through-holes extending between the surfaces. The surfaces have different chemical properties relative to the walls of the through-holes, such that the walls and surfaces can be independently functionalized. For example, the walls can be coated with gold (e.g., by coating the entire platen, including both the walls and the opposing surfaces with gold, and then electropolishing the surfaces to remove the gold therefrom), allowing the walls to be rendered hydrophobic upon treatment with alkane thiols. Conversely, the surfaces (but not the walls) could be coated with metal oxides so that alkanephosphates can be bound thereto.
In another embodiment, the invention features a method of making a plastic platen of a desired thickness, having through-holes. The method features the steps of: a) potting a plurality of capillaries (e.g., glass or plastic capillaries) in the through-holes of a stack of platens comprising at least two platens having through holes; b) separating adjacent platens by a distance equal to the desired thickness; c) injecting a plastic-forming material into the space between the separated platens; d) forming (e.g., heat-setting or curing) the plastic; and e) slicing at the interface between the platens and the plastic to form the chips. The plastic-forming material can be, for example, a photo-, thermo-, or chemical-curable material such as a UV-curable material, e.g., polymethylmethacrylate (PMMA), polystyrene, or epoxy, and the forming step can entail exposing the material to ultraviolet light; or the plastic-forming material can be a molten thermoplastic material and the forming step can involve cooling the material.
In still another embodiment, the invention features a method of making a plastic chip of a desired thickness, having through-holes. The method features the steps of: a) potting a plurality of fibers or wires in the through-holes of a stack of platens comprising at least two platens having through holes; b) separating adjacent platens by a distance equal to the desired thickness; c) injecting a plastic-forming material into the space between the separated platens; d) forming the plastic; e) withdrawing the fibers or wires from the plastic to form through-holes; and f) slicing at the interface between the platens and the plastic to form the chips.
Still another embodiment of the invention is a method of creating a chemical array. The method includes the steps of: a) providing a platen having a plurality of through-holes and two opposing surfaces; b) applying a mask to one or both surfaces of the platen to block at least some of the through-holes, while leaving other through-holes open; c) exposing a surface of the platen to a reagent (e.g., e.g., a liquid, a gas, a solid, a powder, a gel, a solution, a suspension such as a slurry, a cell culture, a virus preparation, or electromagnetic radiation; e.g., by spraying the platen with a solution or suspension of the reagent, or by condensing, pouring, depositing, or dipping the reagent onto the platen) so that the reagent enters at least one of the open through-holes; and d) repeating steps b) and c) (e.g., at least once, generally at least three times; for creation of nucleic acid arrays, the steps can be repeated four times the length of the desired nucleic acid chains; for creation of protein arrays, the steps can be repeated twenty times the length of the desired peptide chains) with at least one different mask and at least one different reagent to create a chemical array. The masks can be reusable or disposable, and can be applied mechanically (e.g., robotically) or manually. The mask can, in some cases, initially include the reagent (e.g., absorbed onto or contained within it). The mask can be flexible or rigid, for example, and can be made of a polymer, an elastomer, paper, glass, or a semiconductor material. The mask can, for example, include mechanical valves, pin arrays (e.g., posts, pistons, tubes, plugs, or pins), or gas jets. In some cases, the xe2x80x9capplyingxe2x80x9d step forms a hermetic seal between the mask and the platen. The mask can also be translated (e.g., moved between the repetitions of the method) to expose different through-holes. In some cases, the mask has co-registration pins and holes such that alignment of pins and holes in the mask register with the through-holes in the platen. In these cases, multiple masks can be made part of a flexible tape, and the multiple masks are registered with the through-holes of the platen by advancing the tape (e.g., the masks can be on a spool, ribbon, or roll, and can be advanced in a manner analogous to the advancing of film in a camera). Arrays created by any of these methods are also considered to be an aspect of the invention.
In yet another embodiment, the invention features a method of creating a chemical array. The method includes the steps of: a) providing a platen having a plurality of through-holes and two opposing surfaces; b) applying a mask that has one or more reagents on its surface to one or both surfaces of the platen to transfer the reagent from the mask to at least some of the through-holes; and c) repeating step b) with at least one different mask and at least one different reagent to create a chemical array.
The invention also features a method for separating samples within a chemical array in a platen. The method includes the steps of a) providing a platen having a plurality of through-holes and two opposing surfaces; b) electrophoretically transporting a charged reagent into at least some of the through-holes by placing the platen into an electrophoresis apparatus containing the reagent and applying an electric field parallel to the through-holes; and c) repeating step b) with at least one different reagent to create a chemical array.
In still another embodiment, the invention features a method of creating a spatially addressable array. The method includes the following steps: a) providing a platen having a spatially addressable plurality of discrete through-holes each having an inner wall, wherein said platen has opposing hydrophobic surfaces; and b) covalently or non-covalently immobilizing at least one reagent or probe on the inner walls of at least some of the through-holes or on a bead contained within at least one of the through-holes to form a spatially addressable array. In this method, the through-holes can be either non-communicating (i.e., the contents of adjacent through-holes do not mix with each other) or selectively communicating (i.e., the walls of at least some of the through-holes act as semi-permeable membranes) through-holes. In some cases, the method can also include the step of: c) flowing reagents (e.g., monomers, wash solutions, catalysts, terminators, denaturants, activators, polymers, cells, buffer solutions, luminescent and chromatogenic substrate solutions, beads, heated or cooled liquids or gases, labelled compounds, or reactive organic molecules) into or through a predetermined subset of the through holes.
Yet another embodiment of the invention is a method of creating a stochastic array. The method includes a) providing a platen having a plurality of through-holes; and b) applying each of a plurality of reagents to the through-holes in a random or semi-random manner (e.g., spatially random or random with respect to distribution of reagents) to create a stochastic array. The xe2x80x9capplyingxe2x80x9d step can include, for example, providing a plurality of dispensing devices addressing at least some of the through-holes, dispensing different combinations of reagent solutions (e.g., as solutions, neat, or in suspension) into each through-hole, and repositioning the dispensing devices at least once to address a different set of through-holes. In this case, the method can also involve dispensing a fluid that is immiscible with the reagent solutions into at least one through-hole.
In another embodiment, the invention features a method of identifying combinations of reagents having a biological, chemical or physical property of interest. The method involves, for example, the use of radiolabelled probes, or the measurement of chemiluminescence. The method features the steps of: a) creating a stochastic array using the above method; b) assaying the stochastic array for combinations having a property of interest; and c) identifying the reagents that have the property of interest. Non-limiting examples of properties of interest include catalysis (see, e.g., Weinberg et al., Current Opinion in Solid State and Materials Science, 3:104-110 (1998)); binding affinity for a particular molecule (see, e.g., Brandts et al., American Laboratory 22:3041 (1990); or Weber et al., J. Am. Chem. Soc. 16:2717-2724 (1994)); ability to inhibit particular chemical and biochemical reactions; thermal stability (see, e.g., Pantaliano et al., U.S. Pat. Nos. 6,036,920 and 6,020,141); luminescence (see, e.g., Danielson et al., Nature 389:944-948 (1997)); crystal structure (see, e.g., Hindeleh et al., Journal of Materials Science 26:5127-5133 (1991)); crystal growth rate; diastereoselectivity (see, e.g., Burgess et al., Angew. Chem. 180:192-194 (1996)); crystal quality or polymorphism; surface tension; (see, e.g., Erbil, J. Phys. Chem. B., 102:9234-9238 (1998)); surface energy (see, e.g., Leslot et al., Phys. Rev. Lett. 65:599-602 (1990)); electromagnetic properties (see, e.g., Briceno et al., Science 270:273-275 (1995); or Xiang et al., Science 268:1738-1740 (1995)); electrochemical properties (see, e.g., Mallouk et al., Extended Abstracts; Fuel Cell seminar: Orlando, Fla., 686-689 (1996)); and optical properties (see, e.g., Levy et al., Advanced Materials 7:120-129 (1995)); toxicity, antibiotic activity, binding, and other biological properties; fluorescence and other optical properties; and pH, mass, binding affinity, and other chemical and physical properties.
In another embodiment yet, the invention features a method of loading a platen having a plurality of through-holes, where the platen has opposing surfaces (e.g., the surfaces are hydrophobic and the through-holes have hydrophilic walls). The method includes the steps of: a) dipping the platen into a liquid sample (e.g., a neat liquid, a solution, a suspensions, or a cell culture) that includes a sample to be loaded into the through-holes, thereby loading at least some of the through-holes with the sample; and b) passing the platen through a liquid that has an affinity for the surfaces of the platen but that is immiscible with the liquid sample, thereby cleaning the surface of the platen of excess sample mixture (e.g., by adding, on top of the sample mixture, the immiscible liquid, where the liquid has a lower density than the sample mixture (e.g., mineral oil); and removing the platen from the sample mixture through the liquid; device comprising a barrier between the sample and the liquid).
The invention also features another method of loading a platen having a plurality of through-holes, where the platen has opposing surfaces. The method includes: a) dipping the platen into a liquid sample comprising a sample to be loaded into the through-holes, thereby loading at least some of the through-holes with the sample; and b) contacting the platen with a liquid that has an affinity for the surfaces of the platen but is immiscible with the liquid sample, thereby cleaning the surface of the platen of excess sample mixture.
The invention also features a method of maintaining the viability of an aerobic organism in a platen having a plurality of through-holes. The method includes the steps of: a) loading the aerobic organism (e.g., a cell or an embryo) into at least some of the through-holes of the platen, and b) submerging the platen into a gas permeable liquid. The organism can be, for example, in a fluid such as a growth medium, in which case the gas permeable liquid should be immiscible with the fluid. The method can also include assaying one or more physical properties of the aerobic organism. The gas permeable liquid can be, for example, a fluorocarbon such as perfluorodecalin, a silicone polymer, or a monolayer (e.g., a monolayer of a lipid or high molecular weight alcohol.
In another embodiment still, the invention features a method of mixing volatile samples with other samples (whether volatile or non-volatile). The method include the steps of: a) providing a platen having a plurality of through-holes; b) optionally loading some or all of the through-holes with one or more non-volatile samples (if any); c) loading at least some of the through-holes of the platen with one or more volatile samples to allow the samples in each through-hole to mix with other samples in the same through-hole; and d) submerging the platen in a liquid immiscible with the volatile samples, where steps b), c) and d) can be performed in any order. In preferred embodiments, step d) is performed prior to introduction of volatile samples. The samples to be mixed can be initially provided in two separate platens that are contacted while submerged in said immiscible liquid to allow mixing. The immiscible liquid can be, for example, a fluorocarbon, a silicone polymer, mineral oil, or an alkane.
The invention also features a method of mixing an array of samples. The method entails: a) providing a platen having a plurality of through-holes, wherein at least some of the through holes are loaded with a first sample or set of samples; b) providing a substantially flat surface comprising an array of a second sample or set of samples, wherein the second sample or set of samples on the flat surface can be registered (e.g., the second sample or set of samples can be arranged in a spatial pattern that allows it to line up with at least some of the through-holes of the platen) with the sample in the platen; c) registering the platen with the array of the second sample or set of samples on the flat surface; and d) contacting the platen with the flat surface, wherein the sample in the platen is aligned with the sample on the flat surface. This method can be used, for example, to avoid cross-contamination; also, registering and contacting can be done simultaneously. In some cases, either the first or second sample or set of samples can include one or more probes. The method can also include the further step of analyzing a physical property (such as fluorescence or other optical properties, pH, mass, binding affinity; e.g., using radiolabelled probes and film, chemiluminescence) of a sample contained in the platen. In some cases, the flat surface can also include a hydrophobic pattern matching the pattern of the platen array (e.g., to prevent cross-contamination).
In another embodiment, the invention features a method for transferring a reagent or probe to a receptacle (e.g., into a bottle, a tube, another platen, a microtiter plate, or a can) from a specific through-hole of a platen comprising a plurality of through-holes. The method includes the steps of: a) placing the platen over the receptacle; and b) applying a burst of gas, liquid, solid, or a pin (e.g., a piston, a tube, a post, a plug) to the specific through-hole to transfer the reagent or probe into the receptacle. The burst of gas, liquid, or solid can be generated, for example, with a syringe, or by depositing a photodynamic or photothermal material (carbon black, plastic explosives, water droplets) in or above the through-hole, and then exposing the photodynamic or photothermal material to a laser beam of frequency and intensity suitable to activate the photodynamic or photothermal material.
In another embodiment, the invention features a device for filling or draining through-holes in a platen having a plurality of through-holes. The device includes: a) a holder adapted to accept the platen; b) a nozzle having an aperture of a suitable size to inject a sample into a single through-hole in said platen; and c) a valve that controls a flow of a sample through said nozzle, wherein the holder and nozzle can move with respect to each other. The nozzle can be, for example, positioned so as to contact the platen (or not). The device can optionally include a microplate (e.g., a microtiter plate) positioned to receive samples from the platen, as well as a computer that can control the valve and control the positions of the holder and nozzle (and, optionally, the microplate) relative to one other. The optional microplate, the holder, and the nozzle can, in some cases, be moved independently of each other in at least two dimensions. Alternatively, the nozzle can be held in a single position while the holder and nozzle can be moved independently of each other in at least two dimensions.
In another embodiment, the invention features a method of analyzing the kinetics of one or more reactions occurring in at least one of the through holes of a platen. The method includes: a) providing a first platen having a plurality of through-holes, wherein the through-holes are loaded with a first sample or set of samples; b) introducing the platen into a detection device; c) introducing a second platen having a plurality of through-holes into the detection device, wherein the through holes are loaded with sample or reagent; d) registering and contacting the platens such that contents of the through-holes of said first platen can mix with contents of corresponding through-holes of said second platen; and e) detecting a change in a physical property of the contents of at least some of the through-holes over time.
In another embodiment, the invention features a method of analyzing a physical property of a sample in an array. The method includes the steps of: a) providing a platen having a plurality of through-holes, where the through-holes are loaded with a sample; b) placing the platen between two partially transmitting mirrors; c) illuminating the samples through one of the mirrors (e.g., with a laser, atomic lamp, or other light source, including white light sources); and d) detecting optical output from the sample. Optionally, mirrors that reflect at only one wavelength and transmit at all others can be used, and non-linear optical effects can also be observed. The xe2x80x9cimagingxe2x80x9d step can involve, for example, measuring light emanating from the array or measuring light emitted from the mirror opposite from the illumination source. The platen can also be placed within a laser cavity, and an optical gain medium can be positioned between the two mirrors.
The invention also features a method of measuring sample output from an array. The method includes the steps of: a) providing a platen having a plurality of through-holes, wherein the through-holes are loaded with sample; b) introducing the sample into an array of capillaries; c) eluting the samples through the capillaries using pulse pressure, creating a non-continuous flow; d) spotting the eluting samples onto a surface that is moving relative to the capillaries (e.g., a web, a tape, a belt, or a film), wherein the spots are discrete and no mixing of the samples occurs; and e) analyzing a physical property of the spots.
The invention also features a method of storing a plurality of samples in an assay-ready, high-density format. The method includes the steps of a) providing a platen having a plurality of through-holes; b) loading the through-holes with the samples (e.g., small molecules) dissolved in a mixture comprising two solvents, a first solvent having a low vapor pressure (e.g., dimethyl sulfoxide (DMSO)) and a second solvent having a higher vapor pressure relative to the first solvent (e.g., ethanol; preferably, both solvents are inert and are able to dissolve the sample); and c) evaporating the second solvent to result in a plurality of samples in first solvent (preferably as films on the walls of the through-holes). The volume of the first solvent in each solution can be, for example, less than about 25 nl (e.g., less than 10 nl, 1 nl, 250 pl, 100 pl, or even less than about 25 pl; e.g., a xe2x80x9cmicrodropletxe2x80x9d). In some embodiments, the sample dissolved in the first solvent forms a film on the wall of a through-hole.
The invention features a method of forming a high throughput assay. The method includes: a) providing a platen having a plurality of through-holes, wherein at least some of the through-holes contain a sample dissolved in a solvent having a low vapor pressure (such as a array of samples prepared for storage according to the above method); b) cooling the platen to a temperature sufficient to freeze the dissolved sample, c) dipping the platen into a solution comprising a reagent, wherein the temperature of the solution is less than the freezing point of the sample, but greater than the freezing point of the reagent solution, d) removing the platen from the reagent solution, and e) warming the platen to a temperature greater than the freezing point of the sample. The reagent solution can be, for example, an aqueous solution.
Yet another embodiment of the invention features a filtration device, having first and second platens, each having a plurality of through-holes, and a semi-permeable membrane. The platens are aligned such that the through-holes of the first platen are substantially aligned with the through-holes of the second platen and the membrane is sandwiched in between the two platens. Optionally, the platens can have hydrophobic surfaces. The semi-permeable membrane can be, for example, a nitrocellulose membrane, or can include a layer of cells.
A xe2x80x9cspatially addressable through-holexe2x80x9d has a position and dimensions that are known to a high degree of certainty (e.g., relative to a reference position on the device). The degree of certainty is sufficient that the through-holes of two platens placed one on top of the other can align, allowing reagents to transfer in a parallel fashion. The degree of certainty is also sufficient such that a sample in any given through-hole can be retrieved by a robotic device that knows only the position in which that hole should be found relative to a reference point on the device. The term xe2x80x9cplanar array of through-holesxe2x80x9d refers to an array of through-holes on a platen such as that described in PCT application WO99/34920.
A xe2x80x9creagentxe2x80x9d is a chemical compound, a gas, a liquid, a solid, a powder, a solution, a gel, a bead, or electromagnetic radiation.
The term xe2x80x9cprobexe2x80x9d or xe2x80x9cchemical probexe2x80x9d refers to a chemical, biological, mechanical, or electronic structure that detects a specific analyte by a specific binding or catalytic event. The binding or catalytic event can be transduced into a signal readable by an operator. One type of chemical probe is an affinity probe (e.g., a specific nucleic acid that binds to another nucleic acid). Examples of mechanical probes include a cantilever that has a ligand immobilized on its surface and a material whose properties (e.g., strain, inertia, surface tension) change in response to a chemical or biological event.
The term xe2x80x9cchemical detection eventxe2x80x9d refers to a chemical reaction between molecule(s) of interest and probe molecule(s) that in turn produces a signal that can be observed by an operator. For example, the hydrolysis of fluorescein di-xcex2-galactoside by the enzyme xcex2-galactosidase, to produce the fluorescent molecule fluorescein, is a chemical detection event. In some cases, the chemical detection event can involve a series of chemical reactions triggered by an initial interaction of analyte and probe (e.g., activation of a signal transduction pathway in a probe cell by the binding of a ligand to a surface receptor).
The term xe2x80x9clinker moleculexe2x80x9d means a molecule that has a high affinity for or covalently links to the surface of a platen or bead. The linker molecule can have a spacer segment such as a carbon chain, and can also have a functional group at its end to enable attachment of probe molecules covalently or with high affinity.
The term xe2x80x9cimmobilizedxe2x80x9d means substantially attached at the molecular level (i.e., through a covalent or non-covalent bond or interaction).
The term xe2x80x9cphotocleavable compoundxe2x80x9d refers to a compound that contains a moiety that, when exposed to light, dissociates into multiple independent molecules.
The term xe2x80x9csmall moleculexe2x80x9d refers to a molecule having a mass less than about 3000 daltons.
The term xe2x80x9chybridizationxe2x80x9d refers to complementary, specific binding of two or more molecules (e.g., nucleic acids) to one another.
xe2x80x9cSolid phase synthesisxe2x80x9d refers to a chemical synthesis process in which at least one of the starting materials in the synthesis reaction is attached to a solid material such as a polymer bead, a gelatinous resin, a porous solid, or a planar surface.
The term xe2x80x9cblotterxe2x80x9d refers to a material capable of capturing excess liquids by absorption.
The term xe2x80x9cbeadxe2x80x9d means a small particle, generally less than about 1 mm (e.g., less than about 100 xcexcm) in any dimension, with the ability to have reagents attached to its surface or stored in its interior. A bead can be made from one or more of a variety of materials, including organic polymers, glass, and metals. The reagent is typically attached to the bead by chemical reaction with a reactive functional group such as a carboxyl, silanol, or amino group on its surface. Reagents can, for example, be confined to the bead by covalent chemical attachment or by physical adsorption to the bead surface. The bead shape can be nearly spherical, irregularly shaped, or of an intermediate shape.
The term xe2x80x9cstringencyxe2x80x9d refers to the degree to which non-specific molecular interactions are disrupted during a washing step.
The term xe2x80x9celectrophoretic washingxe2x80x9d refers to the removal of non-specifically bound, ionic molecules from a probe by applying an electric field.
xe2x80x9cSpecific interactionsxe2x80x9d are interactions between two molecules resulting from a unique three-dimensional structure of at least one of the molecules involved. For example, enzymes have specific interactions with transition state analogues due to their evolution toward stabilizing reaction intermediates.
The term xe2x80x9cmicro-platexe2x80x9d refers to a collection plate used to transfer the contents of the through-holes of an array, where no cross contamination of the through-holes occurs in the transfer.
The term xe2x80x9cmicro-dropletxe2x80x9d means a drop of liquid having a volume of 50 nl or less (e.g., less than about 50 nl, 25 nl, 10 nl, 5 nl, 1 nl, 500 pl, 250 pl, 100 pl, 50 pl, or less).
The term xe2x80x9cphysical propertiesxe2x80x9d means any measurable property of an object or system, including electrical, magnetic, optical, thermal, mechanical, biological, nuclear, and chemical properties.
The new methods have numerous advantages. For example, the new methods allow optimization of processes in a parallel manner. For instance, synthesis of a particular molecular species often requires tedious quantitative investigation of different synthetic methods with a view towards optimizing product yield. Using the new methods, process parameters can be varied on a through-hole-by-through-hole basis in the array, and the product analyzed to determine the protocol best suited for high yield synthesis.
Another advantage of the invention over conventional arrays of chemical probes on a planar substrate is that each chemical detection event takes place in a physically isolated container (i.e., the through-hole), allowing amplification of the signal by catalysis (e.g., releasing detectable molecules into the solution contained in each through-hole). Such detectable molecules include, for example, fluorescent products of a fluorogenic enzyme substrate, and chromogenic products of a chromogenic substrate. Physical isolation of samples retained in the array also prevents cross-contamination by eliminating lateral communication between the through-holes.
Another advantage of the invention is that each through-hole can have a precise and known spatial location in the array. Each through-hole is then spatially addressable, thereby facilitating the insertion and removal of liquids from each through-hole, the analysis of the contents of each through-hole, and the alignment of multiple arrays for highly parallel transfer of reagents.
Another advantage of the invention is that the relative volumes of the members of two arrays can be easily adjusted by changing the depth of one array with respect to the other.
Still another advantage of the invention is that substances that bind to chemical probes contained in the through-hole array can easily be recovered as distinct samples for further analysis. For example, the bound contents of the well can be eluted onto a planar substrate for analysis by matrix-assisted laser desorption and ionization (MALDI) or surface-enhanced laser desorption and ionization (SELDI) mass spectrometry, or nuclear magnetic resonance (NMR) spectroscopy. Alternatively, the contents of the through-hole can be electro sprayed directly from the through-hole into a mass spectrometer. The contents of the through-hole can also be crystallized and analyzed with x-ray or electron diffraction techniques (e.g., to determine crystal structure). This aspect of the invention allows for sensitive detection of unlabelled analytes.
Yet another advantage of the invention is that the samples can be introduced or removed from the platen by electrophoresis, as the through-holes can allow for conduction of an electric field.
Another advantage of the invention is that samples are accessible from both sides of the platens. This means, for example, that samples can be removed from the platens by applying pressure, an air or gas stream, or an explosive charge to a through-hole of interest and then collecting the material from the opposing face of the platen. Alternately, samples can be sucked out of the platen without creating a vacuum. Thus, the volume of the samples in not limited by the current state-of-the-art microfluidics techniques, and a minimum quantity of fluid is lost upon the collection of the sample. A pressure can be applied, for example, in the form of a solid pin (acting, e.g., as a piston), or in the form of a burst of inert gas. Another implication of this advantage is that it is relatively easy to perform electrospray ionization mass spectrometry directly from the platen. Simultaneous measurement of luminescence from two spectrally distinct luminescent probes located in the microchannel array can be performed in either a trans- or epi-illumination optical configuration, including, for example, a light source, an optical filter, and a CCD camera. Optical signals can be collected from both sides of the platen simultaneously.
The numerous samples contained in the platen can be rapidly transferred to a flat surface or membrane, facilitating processes such as SELDI mass spectrometric analysis and growth of bacterial cells (e.g., cells contained in the through-holes), to form individual colonies for storage and further analysis. Transfer from a planar material to the array can also be accomplished, as in electroblotting from a polyacrylamide 2-D protein gel into the array.
Advantageously, the surface area of the liquid exposed to the environment is minimized by the high aspect ratio geometry, thus limiting evaporation.
Still another advantage of the new methods is that the sample contained in a given through-hole constitutes a small thermal mass and can, therefore, reach thermal equilibrium quickly and uniformly. The fact is relevant, for example, to synthetic methods that involve heating and/or cooling steps (e.g., replication of nucleic acids using the polymerase chain reaction, PCR).
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.